Chemical synthesis of reagents for peptide coupling

ABSTRACT

The present invention provides improved methods for synthesis of phosphinothiol reagents, as well as novel protected reagents, for use in formation of amide bonds, and particularly, for peptide ligation. The invention provides phosphine-borane complexes useful as reagents in the formation of amide bonds, particularly for the formation of an amide bond between any two of an amino acid, a peptide, or a protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.10/456,988, filed Jun. 6, 2003, now U.S. pat. 6,974,884, which takespriority under 35 U.S.C. 119(e) from U.S. provisional application Ser.No. 60/387,171, filed Jun. 7, 2002, both of which are incorporated byreference herein to the extent that it is not inconsistent with thedisclosure herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. Government support under NationalInstitutes of Health Grant No. GM44783. The Government has certainrights in this invention.

BACKGROUND

The chemoselective ligation of peptides can be used to effect the totalchemical synthesis of proteins.¹ The most common ligation method, nativechemical ligation, relies on the presence of a cysteine residue at theN-terminus of each ligation junction.^(2,3) Recently, we have reported⁴a peptide ligation method, a “Staudinger Ligation,” that is universal,i.e., independent of the presence of any particular side chain Thismethod is based on the Staudinger reaction, wherein a phosphine reducesan azide via a stable iminophosphorane intermediate.⁵ Acylation of thisiminophosphorane yields an amide.^(6,7)

Scheme 1 illustrates our Staudinger Ligation which is further describedin PCT application PCT/01/15440, filed May 11, 2000. A peptide fragmenthaving a C-terminal phosphinothioester (2) reacts with another peptidefragment having an N-terminal azide (3). The resulting iminophosphorane(4) leads, after an S- to N-acyl shift, to an amidophosphonium salt (5).The P—N bond of the amidophosphonium salt is hydrolyzed readily toproduce the amide product (6) and a phosphine oxide (7). Importantly, noresidual atoms remain in the amide product.^(4,6b), so the ligation istraceless. The phosphinothioester (2) is prepared by reaction of a ofphosphinothiol reagent (1), such as Ph₂CH₂—SH, where Ph is a phenylgroup. The Staudinger Ligation can generally be employed to form peptidebonds and as such can be employed to ligate two amino acids, a peptideor a protein with an amino acid or peptide or two proteins. Moregenerally, the Staudinger Ligation can be employed to form amide bonds.The amide bond is formed between a thioester and an azide. In general,the reaction functions for any thioester and any azide. The thioester isconverted into a phosphinothioester which then reacts with the azide.For example, the thioester group may be formed, at the carboxy group ofan amino acid or at the carboxy terminus of a peptide or protein or atan acid side group of an amino acid or one or more amino acids in apeptide or protein. The azido group may be formed, for example, at theamino group of an amino acid or at the amino terminus of a peptide orprotein or at a basic side group of an amino acid or one or more aminoacids in a peptide or protein. The Staudinger Ligation may also beemployed to ligate an amino acid, peptide or protein group to acarbohydrate group, which may be a mono-, di-, tri- or polysaccharide,or to a nucleoside. The Staudinger Ligation may also be employed toligate an amino acid, a peptide or protein group to a reporter group,tag or label (e.g., a group whose presence can be detected by optical ormass spectrometry or other instrumental method), including a fluorescentor phosphorescent group, an isotopic label or a radiolabel.

All natural α-amino acids except glycine have a stereogenic center attheir α-carbon.⁸ To be an effective tool for the total chemicalsynthesis of proteins, a peptide ligation reaction must proceed withoutepimerization. The coupling of thioesters in native chemical ligation,which like the Staudinger Ligation (Scheme 1) involvestransthioesterification followed by an S- to N-acyl shift,^(2,3) isknown to proceed without detectable racemization.⁹ We have demonstratedthat the Staudinger Ligation (Scheme 1) proceeds in near quantitativeyield without detectable epimerization.

The Staudinger Ligation of Scheme 1 employs a phosphinothiol reagent(1). Previously reported methods of synthesis of such reagents^(4a, 4b)generally proceed in low yield. Synthesis of the phosphinothiol offormula 1 where R and R′ are phenyl groups requires four syntheticsteps, two of which are problematic, with an overall yield of about 39%.Difficulties can also be encountered in the synthesis of reagents offormula 1 where R and R′ are small alkyl groups, such as ethyl groups,due to instability of the reagent itself. Use of the Staudinger Ligationfor the formation of amide bonds between a variety of species would befacilitated by the development of improved methods for the synthesis ofphosphinothiol reagents and the development of such reagents withincreased stability. This invention provides improvements for carryingout the Staudinger Ligation.

SUMMARY OF THE INVENTION

The present invention provides improved methods for synthesis ofphosphinothiol reagents, as well as novel protected reagents, for use information of amide bonds, and particularly for peptide ligation asexemplified in Scheme 1 and Scheme 2.

In a specific embodiment, the invention provides improved methods forsynthesis of phosphinothiols, e.g., 1 (Scheme 1), which were the mosteffective known phosphinothiols for effecting the Staudinger Ligation ofpeptides. In one aspect, the invention provides a synthesis ofphosphinothiols themselves. In another aspect, the invention provides aprotected phosphinothiol reagent (10, Scheme 2) which arephosphine-borane complexes which can be employed in the StaudingerLigation to prepare phosphinothioesters.

A phosphinothiol reagent of this invention (1) is synthesized asillustrated in generalized Scheme 3 by reacting a protected alkylatingagent of formula (20) where PR is a protecting group, particularly anacyl group —COR⁵ (defined below) and X is a leaving group (LG) with aphosphine-borane complex of formula (25, where R, R′ and R″ are definedas in formula 10, below) on deprotonation of the phosphine-boranecomplex to generate the protected phosphine-borane addition complex(10). The phosphinothiol reagent (1) is generated by disruption of thephosphine-borane complex (10) and removal of the protecting group (PR).

In specific embodiments, the protecting group PR is a —CO—R⁵ where R⁵ isH, an alkyl group, an aryl group or a substituted alkyl or a substitutedaryl group. In specific embodiments, R and R′ are aryl groups,particularly phenyl groups. In specific embodiments, R² and R³ are H oralkyl groups. In specific embodiments, R″ are all hydrogen or are allsmall alkyl groups. In specific embodiments, X is a “good leaving group”as that term is understood in the art and specifically X can be ahalogen, or a OTs, Otf, or OMs group.

Alternatively, the phosphine-borane complex of formula 10 can be used

-   -   The invention also provides phosphine-borane complexes of        formula:

where:

PR is a suitable protecting group, which can include, among others,—CO—R⁵ groups where R⁵ can be selected from H, alkyl or aryl groups orsubstituted alkyl or aryl groups, where the substituents do not affectthe function of PR as a protecting group for reactions illustratedherein;

R and R′, independently of one another, are alkyl or aryl groups orsubstituted alkyl or aryl groups where the substituents do notsignificantly detrimentally affect the reactions as illustrated herein,R and R′ may be the same or different groups, R and R′ may be covalentlylinked to each other;

R″, independently of other R″ in the compound, can be H, an alkyl oraryl group or a substituted alkyl or aryl group where the substituentsdo not significantly negatively affect the formation of thephosphine-borane complex or significantly negatively affect theproperties of B(R″)₃ as a protective groups for the phosphine; all threeof R″ may be the same or each may be different, any two or three of R″may be covalently linked to each other; and

R² and R³, independently of one another, can be selected from H, analkyl group, an aryl group or a substituted alkyl group or a substitutedaryl group, where the substituents do not significantly negativelyaffect the function of the phosphine-borane complex in the StaudingerLigation, particularly as illustrated in Scheme 2; R² and R³ may becovalently linked to each other.

In specific embodiments, the invention provides phosphine-boranecomplexes for use as peptide ligation reagents in which R and R′ arealkyl groups, particularly ethyl groups, propyl groups or butyl groups,or aryl groups, particularly phenyl groups or substituted phenyl groups;R″ are all H or small alkyl (e.g., methyl, ethyl, propyl, butyl groups);R² and R³ are H or small alkyl groups (e.g., methyl, ethyl, propyl,butyl groups) and PR is a —CO—R⁵ group where R⁵ is H, and alkyl group oran aryl group.

In more specific embodiments, the invention provides phosphine-boranecomplexes of formula 10 in which R and R′ are alkyl groups, particularlyethyl groups, propyl groups or butyl groups; R″ are all H or small alkyl(e.g., methyl, ethyl, propyl, butyl groups); R² and R³ are H and PR is a—CO—R⁵ group where R⁵ is H, an alkyl group or an aryl group.

In other specific embodiments, the invention provides phosphine-boranecomplexes of formula 10 in which R and R′ are phenyl groups; R″ are allH or small alkyl (e.g., methyl, ethyl, propyl, butyl groups); R² and R³are H and PR is a —CO—R⁵ groups where R⁵ is H, an alkyl group or an arylgroup.

In another aspect, the invention provides phosphine-borane complexes offormula 12:

where R, R′, R″, R2 and R3 are as defined above and AA is an amino acid,peptide or protein or a fully or partially protected derivative thereof.The AA group can be linked to the thio group of the complex of formula12 by formation of a thioester at the COOH group of the amino acid (i.e.PRNHC(R^(A))COOH→PRNHC(R^(A))CO-S-C(R₂R₃)-⊕PRR′-BR″₃, where PR an amineprotecting group and R^(A) is an amino acid side-group), at the carboxylterminus of a peptide or protein (i.e.,PRNH-peptide-COOH→PRNH-peptide-CO-S-C(R₂R₃)-⊕PRR′-BR″₃₎or at a carboxygroup of an amino acid side group R^(A). Dependent upon where thethioester linkage is formed the AA group can be protected, if needed,with appropriate PR groups at its COOH terminus or at a COOH group on anamino acid side-group. The complex of formula 12 indicates formation ofone thioester linkage, however, in cases in which AA is an amino acidwith a carboxylate on R^(A) or a peptide or protein containing one ormore R^(A) containing one or more carboxylates, multiplephosphine-borane complexes in which two or more carboxyl groups (mostgenerally n) of the amino acid or peptide are ligated to the phosphineborane complex of formula 12 can be formed, as illustrated in formula12d:

where n is the number of thioesters linkages in the complex, n can be 1,2, 3, or more. AA can be any naturally-occurring or syntheticallyprepared amino acid or any naturally-occurring or synthetically-preparedpeptide, protein or protein fragment.

In a specific embodiment, AA is a naturally-occurring (D-, L-, achiralor racemic)amino acid and specifically can be selected from the groupconsisting of any one or more of (D-, L-, achiral or racemic) glycine,alanine, valine, leucine, isoleucine, phenylalanine, serine, methionine,proline, tyrosine, tryptophan, lysine, arginine, histidine, aspartate,glutamate, asparagine, glutamine, cysteine, methionine, hydroxyproline,γ-carboxyglutamate, O-phosphoserine, ornithine, homoarginine and variousprotected derivatives thereof. Amino acid protecting groups can beselected from any of those known in the art including, but not limitedto, Mtr, Pmc, Tos, Mts, Mbh, Tmob, Trt, Xan, tBu, Bzl, OcHEX, Acm,S-tBu, MeBzl, Mob, Bum, Dnp, Bom, Z, CIZ, Boc, CHO or BrZ whereconventional abbreviations have been employed to name protecting groups.Those of ordinary skill can select from among the known amino acidprotecting groups including those specifically listed, a protectinggroups appropriate for a given amino acid and a given moiety within agiven amino acid and for a group that is chemically compatible for usein the reactions of this invention.

In additional specific embodiments, the invention providesphosphine-borane complexes of formula 12d where R and R′ are alkylgroups or aryl groups which may optionally be substituted andparticularly those in which R and R′ are both ethyl groups. In otherspecific embodiments, the invention provides phosphine-borane complexesof formula 12d where R², R³ are H. In other specific embodiments, theinvention provides phosphine-borane complexes of formula 12d where R″are all H or all small alkyl, e.g., methyl or ethyl.

In other specific embodiments, the invention provides phosphine-boranecomplexes of formula 10 in which R and R′ are alkyl groups or phenylgroups; R″ are all H; R² and R³ are H and PR is a —CO—R⁵ groups where R⁵is H, an alkyl group or an aryl group.

The invention further provides kits for the ligation of amino acids,peptides or proteins which comprise one or more phosphine-boranecomplexes of formula 10, 12 in combination with instructions forcarrying out a Staudinger Ligation as illustrated in Scheme 2 or moregenerally for formation of an amide bond between a thioester and anazide. The phosphine-borane reagents of formula 10, 12 can be providedin one or more suitable containers or receptacles in the kit and may bepre-weighed to provide sufficient reagent for conducting a ligationreaction on a selection scale for a selected amount of starting aminoacids, peptide or proteins to be ligated. The kits may additionalcontain one or more protected amino acid starting materials or otherstarting materials for ligation. The kit may further contain one or moresolvents for conducting the reaction, a deprotecting agent fordeprotecting the phosphine-borane complex or other useful reagents ormaterials useful in the purification of starting materials for ligationor end-products of ligation.

The Staudinger Ligation can be employed generally to form amide bonds.The amide bond is formed between a thioester and an azide and thereaction most generally functions, for any thioester and any azide. Inthe reaction, the thioester group may be formed, at the carboxy group ofan amino acid or at the carboxy terminus of a peptide or protein or atan acid side group of an amino acid or one or more amino acids in apeptide or protein. The azido group may be formed, for example, at theamino group of an amino acid or at the amino terminus of a peptide orprotein or at a basic side group of an amino acid or one or more aminoacids in a peptide or protein. The reagents of this invention may alsobe employed to ligate an amino acid, peptide or protein group to acarbohydrate group, which may be a mono-, di-, tri- or polysaccharide,or to a nucleoside. The reagents of this invention may also be employedto ligate an amino acid, a peptide or protein group to a reporter group,tag or label (e.g., a group whose presence can be detected by optical ormass spectrometry or other instrumental method), including a fluorescentor phosphorescent group, an isotopic label or a radiolabel. Theinvention provides kits comprising one or more phosphine-boranecomplexes of formula 10, 12 for formation of an amide bond and morespecifically for ligation of an amino acid, peptide or protein to acarbohydrate, a nucleoside or to a reporter group, tag or label.

This invention also provides an improved method for forming an amidebond by Staudinger Ligation which employees a phosphine-borane reagentof formula 10, 12.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates generally to improved methods for forming amidebonds and more specifically to improved synthetic methods for formingreagents useful in forming amide bonds and improved reagents for formingamide bonds.

The following terms are defined for use herein:

Alkyl groups refer to saturated hydrocarbon groups which may be linear,branched or cyclic. Small alkyl groups are those having from one to sixcarbon atoms. Alkyl groups may be substituted so long as thesubstituents do not significantly detrimentally affect the function ofthe compound or portion of the compound in which it is found.

Aryl groups refer to groups which contain at least one aromatic ringwhich can be a five-member or a six-member ring. The one or more ringsof an aryl group can include fused rings. Aryl groups may be substitutedwith one or more alkyl groups which may be linear, branched or cyclic.Aryl groups may also be substituted at ring positions with substituentsthat do not significantly detrimentally affect the function of thecompound or portion of the compound in which it is found. Substitutedaryl groups also include those having heterocyclic aromatic rings inwhich one or more heteroatoms (e.g., N, O or S, optionally withhydrogens or substituents for proper valence) replace one or morecarbons in the ring.

Scheme 3A provides a generalized method for synthesis of aphosphinothiol reagent (1) of this invention. The synthesis is based onthe reaction of an alkylating agent 20 and a borane-organophosphinecomplex 25. The phosphine of the phosphine-borane complex 25 isdeprotonated with base followed by alkylation with 20 to givephosphine-borane complex 10. Phosphine-borane complexes 10 are stable toair and moisture and can be stored at room temperature for monthswithout any sign of oxidation or decomposition.

The borane complex 10 is disrupted by mild heating, in the presence ofan amine, preferably with DABCO in toluene for 4 hr to generate aprotected phosphinothiol 11 (PR in this case should be resistant todeprotection under the conditions of complex disruption IIIa). Apreferred protecting group is an —CO—R⁵ group, particularly an acylgroup, which can be removed as previously described^(4b) to give thephosphinothiol 1. The use of and methods for removal of other suitableprotecting groups is known in the art.

Useful bases for deprotonation of the phosphine-borane complex 25 in thefirst step of the synthesis of Scheme 3A are NaH, LiH, KH, KOtBu, NaOMe,NaOEt and the use of NaH is preferred. Amine bases are not preferred, asthey can remove the BH₃ protecting group. While DMF is a preferredsolvent for this first step, other useful solvents include THF, toluene,DMA and more generally any solvent that will dissolve the variouscomponents and not significantly detrimentally affect the desiredreaction. Solvents including thiols, thioethers, and amines should notbe used.

The preferred reagent for disrupting the borane complex 10 is DABCO(1,4-diazabicyclo[2.2.2]octane), but primary, secondary, tertiary oraromatic amines, thiols or thioethers can also be used in this step.Amines such as pyridine, N,N,N′,N′-tetra-methylethylenediamine,diethylamine, triethylenediamine, or dimethylsulfide can specifically beused. The preferred solvent for this step is toluene, but benzene, THF,or any solvent that will dissolve the reagents can be used.

The protecting group of the acyl phosphinothiol can be removed byreaction with base in alcohol. Preferred deprotection agent is 1 eq.NaOH in MeOH. Other reagents include excess NaOH, (1 equivalent orexcess) LiOH, KOH, NH₃, NH₂OH NaHCO₃ in H₂O/THF, LiAlH₄ in ether, AgNO₃in MeOH or a lipase enzyme. Methanol is the preferred solvent, but otheralcohols (including EtOH, iPrOH) or H₂O can be employed. Oxygen gasshould be removed from the solvent to prevent oxidation of the phospheneto a phosphene oxide. DMSO should be avoided as a solvent in this step

The protecting group of the acyl phosphinothiol can be removed byreaction with base in alcohol. Preferred deprotection agent is 1 eq.NaOH in MeOH. Other reagents include excess NaOH, (1 equivalent orexcess) LiOH, KOH, NH₃ or NH₂OH. Methanol is the preferred solvent, butother alcohols (including EtOH, iPrOH) or H₂O can be employed. O₂ (g)should be removed from the solvent to prevent oxidation of phosphene toa phosphene oxide. DMSO should be avoided as a solvent in this step.

Scheme 3B illustrates the synthesis of a specific phosphinothiol 1bwhere R and R′ are both phenyl groups and R² and R³ are both hydrogens.The illustrated synthesis of Scheme 3B gave an overall yield of about74%. See the Examples for experimental details.

With respect to starting reagents in Scheme 3A, R and R′ can generallybe any organic moiety (including alkyl and aryl) that does not containan amine, thiol or thioether. The R′ and R groups can be linked to P viaa C—P or O—P bond. More specifically, R′ and R are optionallysubstituted alkyl group alkoxide group, aryl group or aryloxy group.Alkyl groups include straight-chain, branched or cyclic alkyl groups.Aryl groups may contain one or more (preferably one or two) aromaticrings which may be carbocyclic or heterocylic rings. Preferred alkylgroups are optionally substituted ethyl groups. Preferred alkoxy groupsare ethoxy groups. Preferred aryl groups are optionally substitutedphenyl groups including phenyl groups and halogen (particularlyfluorine)-substituted or carboxy-substituted phenyl groups.

Various known protecting groups (PR) can be employed in the startingreagents of Scheme 3A. One or ordinary skill in the art in view of theteachings herein and what is well-known in the art can selectedappropriate protecting groups from those available in the art. Preferredprotecting groups are —CO—R⁵ groups where R5 is hydrogen, alkyl, aryl orsubstituted alkyl or substituted aryl groups (R⁵ can specifically behydrogen, methyl, ethyl or other small alkyl group, a —CH₂—Ph group(Ph=phenyl), a —CH₂—Ph(Y)_(n) group where Y is a substituent and n isthe number of substituents (Y can, for example, be a halogen, includingfluorine, or —OR⁷ where R⁷ is an optionally substituted alkyl or arylgroup.)

In all cases, optional substituents include halogens and alkoxy groupsand for appropriate groups can be alkyl, and or aryl substituents.Optional substituents do not include amines, thiols or thioester groups.

In Scheme 3A in the thioester reagent is a “good leaving group”, as thatterm is generally known and accepted in the art, that does not includean amine, a thiol or a thioether group. Preferred X are halogens (Br, Clor I), OTs (tosyl, CH₃C₆H₄SO₂—), OTf (triflate, CF₃SO₂—), or OMs (mesyl,CH₃SO₂—). The most preferred X is Br.

The leaving group X is preferably separated from S by a —CR²R³— group,e.g., —CH₂— where R² and R³ are both hydrogens, as illustrated inSchemes 3A and 3B. However, the linker to S can also be —CH₂—CH₂— or ano-substituted Phe group as in

where R² and R³ groups, independent of other R² and R³ groups in thesame molecule, are as defined above and where Ar is an optionallysubstituted aryl group which may contain one or more aromatic rings. PRis preferably an acyl group, e.g., a —COR⁵ group as defined above.

Starting materials and reagents for the reactions of Scheme 3A arereadily available either from commercial sources, by use of knownsynthetic methods or by routine adaptation of known synthetic methods.

Scheme 4A provides two related generalized methods for synthesis ofprotected reagents 10 of this invention. The synthesis is based on theaddition of an aldehyde or ketone (31) to a phosphine-borane complex(not specifically shown) to form the phosphine-borane complex alcohol35. The phosphine of the initial phosphine-borane complex formed isdeprotonated with base followed by addition of the aldehyde or ketone togive the derivatized alcohol phosphine-borane complex 35. Thealcohol-containing phosphine-borane complex (35) is activated byintroduction of an activating group A by reaction with AX (37). Thealcohol is activated for reaction with a protected thiol (39), such asan acyl thiol, particularly thioacetic acid to form the phosphine-boranecomplex 10. Complexes 10 are stable to air and moisture and can bestored at room temperature for months without any sign of oxidation ordecomposition.

Alternatively, reaction IVB can be used to directly prepare complex 10by replacing aldhyde or ketone 31 with the sulfur analogs 32.

The borane complex 10 can be disrupted by mild heating, in the presenceof an amine, preferably with DABCO in toluene to generate a protectedphosphinothiol 11 (PR in this case should be resistant to deprotectionunder the conditions of complex disruption). A preferred protectinggroup is an acyl group, e.g., a —CO—R⁵ group, particularly an acetylgroup, which can be removed as described in references 4a and b to givea phosphinothiol of formula 1 which can be employed as illustrated inScheme 3A in the Staudinger Ligation to form a thioester. The use of andmethods for removal of other suitable protecting groups is known in theart.

However, rather than generating the phosphinothiol 1, complex 10 can beused to generate derivatized amino acid, peptide or protein reagents offormula 12, which can be employed in the Staudinger Ligation asillustrated in Scheme 2. The complex of formula 10 is coupled with anamino acid, peptide or protein that is activated by incorporation of agood leaving group LG. The amino acid, peptide or protein of the complexof formula 12 is provided with appropriate protecting groups to allowthe reaction to proceed as indicated in the first reaction of Scheme 2.It has been found that derivatized amino acid, peptide or proteincomplexes of formula 12, on disruption of the phosphine-borane complex,react with azides as indicated in Scheme 2. Scheme 2 illustrates theStaudinger Ligation to form a n amide bond between two peptides.Complexes of formula 12d can also be employed to form amide bondsbetween any two of an amino acid, peptide or protein. Complexes offormula 12d can further be employed to form an amide bond between anamino acid, a peptide or a protein and a carbohydrate, a nucleoside or asuitable reporter, tag or label.

The method of Scheme 2 is preferred for use with phosphinothiols thatare unstable, for example those in which R and R′ are ethyl groups.

Amine bases are not preferred, for use in the reactions of Schemes 2 and4A, unless otherwise stated, as they can remove the BH₃ protectinggroup. In additional to the preferred base KOH, for formation ofcomplexes 35 and 36, other bases including NaH, LiH, KH, KOtBu, NaOMe,and NaOEt can be used. While THF is a preferred solvent for thisreaction, other useful solvents include THF, toluene, DMA and moregenerally any solvent that will dissolve the various components and notsignificantly detrimentally affect the desired reaction. Solventsincluding thiols, thioethers, and amines should not be used in thereactions of Schemes 2 and 4A, unless otherwise indicated.

The preferred reagent for disrupting the phosphine-borane complexes isDABCO (1,4-diazabicyclo[2.2.2]octane), but primary, secondary, tertiaryor aromatic amines, thiols or thioethers can also be used in this step.Amines such as pyridine, N,N,N′,N′-tetra-methylethylenediamine,diethylamine, triethylenediamine, or dimethylsulfide can specifically beused. The preferred solvent for this step is toluene, but benzene, THF,or any solvent that will dissolve the reagents can be used.

The protecting group of the protected complex 10 can be removed, ifdesired, for example by reaction with base in alcohol. Other reagentsinclude excess NaOH, (1 equivalent or excess) LiOH, KOH, NH₃, NH₂OHNaHCO₃ in H₂O/THF, LiAlH₄ in ether, AgNO₃ in MeOH or a lipase enzyme.Oxygen gas should be removed from the solvent to prevent oxidation ofthe phosphene to a phosphene oxide.

Scheme 4B illustrates the synthesis of a specific phosphine-boranecomplex 10c where R and R′ are both ethyl groups and R² and R³ are bothhydrogens.

As noted above complexes of formula 12 and 12d can be employed asillustrated, for example, in Scheme 2 to form amide bonds between aminoacids, peptides or proteins or between an amino acid and anotherspecies, such as a carbohydrate (e.g., a saccharide) a nucleoside orsimply to an appropriate reporter group, tag or label. If desired, thecomplex 12 or 12d can be disrupted as illustrated in Scheme 2 (page 2)employing DABCO or other amine.

Starting materials and reagents for the reactions of Scheme 4A arereadily available either from commercial sources, by known syntheticmethods or routine adaptation of known synthetic methods.

Several non-glycyl α-azido acids were prepared to examine epimerizationduring the Staudinger ligation. The azido benzamides of both the D and Lenantiomers of phenylalanine, serine, and aspartic acid were prepared(Scheme 5). The azido group was prepared by diazo transfer;¹⁰ thebenazmide was prepared by DCC/HOBt coupling with benzyl amine.Phenylalanine, aspartic acid, and serine were chosen as beingrepresentative of three distinct side chains and moderate(phenylalanine) to high (aspartate and serine) propensity to epimerizeduring standard peptide couplings.¹⁶

Each of these azido acids was coupled with phosphinothioester 51 (whichis AcGlySCH₂PPh₂;Table 1). The couplings were carried out in THF/H₂O(3:1) for 12 h at room temperature with a 1:1 stoichiometry of startingmaterials. The resulting peptides were purified by flash chromatographyto give a nearly quantitative yield of each product (Table 1). The highyield of this equimolar reaction of phosphinothiol 1 with non-glycylazides is consistent with those observed previously^(4b). Thepreparation of phosphinothioester 51 was modified from that described inreferences 4a and b in which coupling using DCC alone led to loweryields and several undesired side products. Pretreatment of N-acetylglycine with HOBt and DCC followed by addition of the phosphinothiol 1improved the yield dramatically. See the Examples.

The chirality of the Staudinger Ligation products from the reaction ofthe D and L α-azido acids was analyzed by HPLC using a D-phenylglycinechiral column. The chromatographic conditions enabled the baselineresolution of the two possible enantiomeric products (FIG. 1). Materialsto be analyzed were injected onto a D-phenylglycine analytical HPLCcolumn and eluted with 30% (v/v) isopropanol in hexanes (isocratic0 for20 min followed by a shallow gradient to 50% (v/v) isopropanol for 40min. After reaction of the D epimer, there was no evidence of productcontaining the L epimer, and vice versa. Thus, the Staudinger Ligationproceeds without detectable epimerization of the α-carbon of the azidoacid. The detection limit of the HPLC chromatographic analysis used isestimated to be ≦0.5%, so that the Staudinger Ligation proceeds with≧99.5% retention of chirality.

Those or ordinary skill in the art will appreciate that startingmaterials, reagents, solvents, temperature and other reaction conditionsother than those specifically disclosed, can be employed in the practiceof this invention without resort to undue experimentation. All suchart-recognized equivalents are included to be encompassed by thisinvention. All references cited herein are incorporated by reference intheir entirety. In particular, published PCT application WO 01/87920 iscited herein and incorporated by reference herein to provide details ofthe Stauding Ligation and method for amide bond formation using thephosphinothiol reagents (1) and the phosphine-borane complexes 10, 12and 12d.

THE EXAMPLES

Amino acids were from NovaBiochem (San Diego, Calif.) and all otherchemicals and solvents were from Aldrich (Milwaukee, Wis.). Reactionswere monitored by thin-layer chromatography using Whatman TLC plates (ALSIL G/UV) with visualization by UV light or staining with ninhydrin orI₂. Silica gel used in flash chromatography was obtained from SiliCycle(Quebec, Canada). Chiral HPLC was performed with a D-phenylglycineanalytical chiral column from MetaChem (Torrance, Calif.). NMR spectrawere obtained with a Varian INOVA-500 MHz spectrometer or a BrukerAC-300 300 MHz spectrometer at the University of Wisconsin nuclearmagnetic resonance facility. Carbon-13 and phosphorus-31 NMR spectrawere both proton-decoupled and phosphorus-31 spectra were referencedagainst an external standard of deuterated phosphoric acid (0 ppm). Massspectra were obtained with electrospray ionization (ESI) techniques on aMicromass LCT instrument.

Borane-thioacetic acid S-[(diphenylphosphanyl)-methyl] ester complex(10b)

Borane-diphenylphosphine complex 25b (10.33 g, 51.6 mmol) was dissolvedin dry DMF under Ar(g) and cooled to 0° C. NaH (1.24 g, 51.6 mmol) wasadded slowly, and the mixture was stirred at 0° C. until bubblingceased. Alkylating agent 20b¹³ (8.73 g, 51.6 mmol) was then added, andthe mixture was allowed to warm to room temperature and stirred for 12h. Solvent was removed under reduced pressure, and the residue waspurified by flash chromatography (silica gel, 10% v/v EtOAc in hexanes).Compound 10b was isolated as a colorless oil in 86% yield. Spectraldata. ¹H NMR (300 MHz, CDCl₃) δ 7.74-7.67 (m, 4H), 7.54-7.41 (m, 6H),3.72 d, J=6 Hz, 2H, 2.23 (s, 3H), 1.51-0.53 (broad m, 3H) ppm; ¹³C NMR(75 MHz, CDCl₃) δ 192.94, 132.26 (d, J=9.2 Hz), 131.61 (d, J=2.3 Hz),128.71 (d, J=10.2 Hz), 127.43 (d, J=55.4 Hz), 29.87, 23.59 (d, J=35.5.Hz) ppm; ³¹P NMR (121 MHz, CDCl₃) 19.40 (d, J=59.3 Hz) ppm; MS (ESI) m/z311.0806 (MNa⁺[C₁₅H₁₈BOPSNa]=311.0807).

Thioacetic acid S-[(diphenylphosphanyl)-methyl] ester (11b). Compound10b (4.00 g, 13.9 mmol) was dissolved in toluene (0.14 L) under Ar(g).DABCO (1.56 g, 13.9 mmol) was added, and the mixture was heated at 40°C. for 4 h. Solvent was removed under reduced pressure, and the residuewas dissolved in CH₂Cl₂ and washed with both 1 N HCl and saturatedbrine. The organic layer was dried over MgSO₄(s), and the solvent wasremoved under reduced pressure. Compound 1b was isolated in 95% yield,and was used without further purification. Spectral Data. As reportedpreviously.^(4b)

(Diphenylphosphino)methanethiol (1b). Compound 11b (17.27 g, 63.0 mmol)was dissolved in anhydrous methanol and Ar(g) was bubbled through thesolution for 1 h. Sodium hydroxide (2.52 g, 63 mmol) was then added, andthe mixture was stirred under Ar(g) for 2 h. Solvent was then removedunder reduced pressure, and the residue was dissolved in methylenechloride. This solution was washed twice with 2 N HCl and once withbrine. The organic layer was dried over MgSO₄(s) and filtered, and thesolvent was removed under reduced pressure. The residue was purified bychromatography (alumina, 25% v/v ethyl acetateinhexanes) to afford 1b asa clear oil in 94% yield. Alternatively, phosphinothiol 1b can be usedin its crude form for formation of phosphinothioesters. Spectral data.¹H NMR (CDCl₃, 300 MHz) δ 7.41-7.38 (m, 4H), 7.33-7.26 (m, 6H), 3.02 (d,J=7.8 Hz, 2H), 1.38 (t, J=7.5 Hz, 1H) ppm; ¹³C NMR (CDCl₃, 75 MHz) δ132.54 (d, J=17.1 Hz), 128.86, 128.36, 128.14, 20.60 (d, J=21.7 Hz) ppm;³¹P NMR (CDCl₃, 121 MHz) 6-7/94 ppm; MS (ESI) m/z 232.05 (MH⁺=233.0,fragments at 183.0, 155.0, 139.0, 91.2).

2(S)-Azido-N-benzyl-3-phenyl-propionamide (48-L). N₃(L)PheOH (1SL) wassynthesized from L-phenylalanine essentially by the procedure ofLundquist and Pelletier.¹⁷ N₃(L)PheOH (1.08 g, 5.7 mmol) was dissolvedin anhydrous DMF (40 mL). HOBt (0.87 g, 5.7 mmol) was then added,followed by DCC (1.17 g, 5.7 mmol). Once precipitate was observed in thereaction, benzyl amine (0.62 mL, 5.7 mmol) was added. The reaction wasallowed to stir under Ar(g) for 3 h. The resulting precipitate (DCU) wasremoved by filtration, and the filtrate was concentrated under reducedpressure to give a yellow oil. This oil was purified by flashchromatography (silica gel, 35% v/v/ethyl acetate in hexanes). N₃(L)Penh(48-L) was isolated as an off-white solid in 90% yield. The procedurewas repeated with D-phenylalanine to give N₃(D)Penh products (D and Lenantiomers) are identical. N₃(L)Penh (48-L) ¹H NMR (500 MHz, CDCl₃ δ7.31-7.12 (m, 8H), 7.11 (m, 2H), 6.55 (bus, 1H), 4.38 (m, 2H), 4.22(did, J=7.8, 4.6 Hz, 1H), 3.34 (did, J=14.0, 4.5 Hz, 1H), 3.07 (did,J=14.1, 7.5 Hz, 1H) ppm; ¹³C NMR (125 MHz, CDCl₃) δ 168.30, 137.35,135.96, 129.51, 128.61, 128.66, 128.61, 128.55, 127.70, 127.68, 127.66,127.57, 127.16, 65.40, 43.41, 38.41 ppm; MS (ESI) m/z 303.1235(MNa⁺[C₁₆H₁₆N₄ONa]=303.1222).

3(S)-Azido-N-benzyl-succinamic acid methyl ester (49-L).Benzyl-protected L-aspartate was used in the procedure of Lundquist andPelletier¹⁷ to give N₃(L)Asp(OMe)OH (46-L). Under these conditions, weobserved transesterification to give the methyl ester product as opposedto the benzyl ester. N₃(L)Asp(OMe)OH (46-L) was produced as a yellowishoil in 78% yield. N₃(L)Asp(OMe)OH (46-L) was then coupled withbenzylamine as above to give N₃(L)Asp(OMe)NHBn (49-L) as a yellowish oilin 90% yield (70% overall, two steps). The procedure above was repeatedwith benzyl-protected D-aspartate to give N₃(L)Asp(OMe)NHBn (49-D) as ayellowish oil in 67% overall yield. Spectral Data. The spectral data forboth N₃Asp(OMe)OH (D and L enantiomers) and both N₃Asp(OMe)NHBn (D and Lenantiomers) products are identical. N₃(L)Asp(OMe)OH (46-L) ¹H NMR (500MHz, CDCl₃) δ 10.24 (bs,1H), 4.47 (dd, J=7.4, 5.3 Hz, 1H), 3.76 (s, 3H),2.91 (dd, J=16.9, 5.1 Hz, 1H), 2.79 (dd, J=16.8, 7.6 Hz) ppm; ¹³C NMR(125 MHz, CDCl₃) δ 174.68, 170.12, 50.09, 52.44, 35.84 ppm; MS (ESI) m/z196.0340 (MNa⁺[C₅H₇N₃O₄Na]=196.0334). N₃(L)Asp(OMe)NHBn (49-L) ¹H NMR(500 MHz, CDCl₃) δ 7.38-7.27 (m, 5H), 6.83 (bs, 1H), 4.54 (m, 3H), 3.75(s, 3H), 3.18 (dd, J=17.1, 3.7 Hz, 1H), 2.75 (dd, J=17.3, 8.7 Hz, 1H)ppm; ¹³C NMR (125 MHz, CDCl₃) δ 170.77, 167.90, 137.35, 128.81, 127.80,127.77, 60.32, 52.24, 43.71, 37.00 ppm; MS (ESI) m/z 285.0953(MNa⁺[C₁₂H₁₂N₄O₃Na]=285.0964).

2(S)-Azido-N-benzyl-3-benzyloxy-propionamide (50-L)

Benzyl-protected L-serine was used in the procedure above to giveN₃(L)Ser(Bzl)NHBn (50-L) as a yellowish oil in 93% yield. The procedurewas repeated with benzyl-protected D-serine to give N₃(D)Ser(Bzl)NHBn(50-D) as a yellowish oil in 90% yield. Spectral data. The spectral datafor both N₃Ser(Bzl)NHBn products (D and L enantiomers) are identical.N₃(L)Ser(Bzl)NHBn (50-L) ¹H NMR (500 MHz, CDCl₃) δ 7.36-7.23 (m, 10H),6.86 (bs, 1H), 4.57 (s, 2H), 4.43 (m, 2H), 4.25 (dd, J=6.9, 3.5 Hz, 1H),4.01 (dd, J=10.3, 3.5 Hz, 1H), 3.83 (10.1, 6.7 Hz, 1H), ppm; ¹³C NMR(125 MHz, CDCl₃) δ 166.81, 137.40, 137.22, 128.65, 128.40, 127.81,127.59, 127.54, 73.45, 70.54, 63.28, 43.38 ppm; MS (ESI) m/z 333.1337(MNa⁺[C₁₇H₁₈N₄O₂Na]=333.1327).

Acetylamino-thioacetic acid S-[(diphenylphosphanyl)-methyl] ester (51).N-Acetylglycine (1.90 g, 16.2 mmol) was dissolved in anhydrous DMF (75mL). HOBt (2.48 g, 16.2 mmol) was added to the resulting solutionfollowed by DCC (3.34 g, 16.2 mmol). Once precipitate (DCU) wasobserved, phosphinothiol 1b was added (3.77 g, 16.2 mmol). The reactionmixture was allowed to stir under Ar(g) for 3 h. The precipitate wasremoved by filtration, and the filtrate was concentrated under reducedpressure to give a white solid. This solid was dissolved in ethylacetate and purified by flash chromatography (silica gel, ethylacetate). Compound 51 was isolated in 96% yield. Spectral Data. Asreported previously

2(S)-(2-Acetylamino-acetylamino)-N-benzyl-3-phenyl-propionamide (52-L).N-Acetylglycylphosphinothioester 9 (0.166 g, 0.5 mmol) and N₃(L)PheNHBn(18-L) (0.140 g, 0.5 mmol) was dissolved in THF/H₂O (3:1, 4 mL), and themixture was stirred at room temperature for 12 h. Solvent was removedunder reduced pressure, and the residue was purified by flashchromatography (silica gel, 5% v/v methanol in dichloromethane).AcGly(L)PheNHBn (52-L) was obtained as a white solid in 90% yield. Theprocedure was repeated with N₃(D)PheNHBn (48-D) to give AcGly(D)PheNHBn(22-D) in 93% yield. Spectral Data. The spectral data for both dipeptideproducts (D and L enantiomers) are identical.

AcGly(L)PheNHBn (52-L) ¹H NMR (300 MHz, CDCl₃:CD₃OD 1:1) δ 7.30-7.22 (m,6H), 7.19-7.16 (m, 2H), 7.16-7.11 (m, 2H), 4.63 (t, J=7.3 Hz, 1H), 4.33(dd, J=31.1, 14.6 Hz, 2H), 3.79 (dd, J=33.1, 16.7 Hz, 2H), 3.12 (dd=J13.8, 7.2 Hz, 1H), 2.98 (dd, J=13.7, 7.2 Hz, 1H), 1.98 (s, 3H) ppm; ¹³CNMR (125 MHz, CDCl₃:CD₃OD 1:1) δ 171.98, 170.93, 169.33, 137.29, 136.00,128.76, 128.05, 127.97, 127.03, 126.75, 126.41, 54.16, 42.79, 42.37,37.52, 21.56 ppm; MS (ESI) m/z 376.1624 (MN⁺[C₂₀H₂₃N₃O₃Na]=376.1637).

3(S)-(2-Acetylamino-acetylamino)-N-benzyl-succinamic acid methyl ester(53-L)

N₃(L)Asp(OMe)NHBn (49-L) was used in the procedure above to giveAcGly(L)Asp(OMe)NHBn (53-L) as a white solid in 91% yield. The procedurewas repeated with N₃(D)Asp(OMe)NHBn (49-D) to give AcGly(D)Asp(OMe)NHBn(53-D) as a white solid in 95% yield. Spectral Data. The spectral datafor both AcGlyAsp(OMe)NHBn products (D and L enantiomers) are identical.AcGly(L)Asp(OMe)NHBn (53-L) ¹H NMR (500 MHz, CDCl₃:CD₃OD 1:1) δ7.34-7.23 (m, 5H), 4.84 (t, J=5.7 Hz, 1H), 4.34 (s, 2H), 3.84 (q, J=16.6Hz, 2H), 3.69 (s, 3H), 2.87 (m, 2H), 2.01 (s, 3H) ppm; ¹³C NMR (125 MHz,CDCl₃:CD₃OD 1:1) δ 177.24, 176.43, 175.18, 174.58, 142.56, 133.17,133.03, 131.93, 131.78, 56.46, 54.13, 47.91, 47.81, 47.67, 40.11, 26.59ppm; MS (ESI) m/z 358.1388 (MNa⁺[C₁₆H₂₁N₃O₅Na]=358.1379).

2(S)-(2-Acetylamino-acetylamino)-N-benzyl-3-benzyloxy-propionamide(54-L)

N₃(L)Ser(Bzl)NHbn (50-L) was used in the procedure above to giveAcGly(L)Ser(Bzl)NHBn (54-L) as a white solid in 92% yield. The procedurewas repeated with N₃(D)PheNHBn (50-D) to give AcGly(D)Ser(Bzl)NHBn(54-D) as a white solid in 99% yield. Spectral Data. The spectral datafor both AcGlySer(Bzl)NHBn products (D and L enantiomers) are identical.AcGly(L)Ser(Bzl)NHBn (54-L) ¹H NMR (500 MHz, CDCl₃:Cd₃OD 1:1) δ7.34-7.21 (m, 10H), 4.60 (t, J=4.4 Hz, 1H), 4.43 (dd, J=23.9, 14.9 Hz,2H), 3.85 (m, 3H), 3.69 (dd, J=9.6,4.6 Hz, 1H), 1.98 (s, 3H) ppm; ¹³CNMR (125 MHz, CDCl₃:CD₃OD 1:1) δ 172.19, 169.86, 169.61, 137.49, 127.87,127.31, 127.23, 127.76, 126.59, 72.88 69.07, 52.93, 42.71, 42.49, 21.38ppm; MS (ESI) m/z 406.1750 (MNa⁺[C₂₁H₂₅N₃O₄Na]=406.1743.

To a solution of borane dimethylsulfide complex (10M in THF, 2.5 mL) infreshly distilled tetrahydrofuran (10 mL) was added diethylphosphine(30b, 2 mL, 17.45 mmol). The solution was stirred under nitrogen for 2hours. The reaction was carefully quenched with ice, and then extractedwith ethyl acetate (3×10 mL). The combined organic layers were washedwith brine (2×10 mL), dried with magnesium sulfate, filtered andconcentrated. The crude phosphine-borane complex was dissolved in amixture of tetrahydrofuran (10 mL) and aqueous formaldehyde (37%, 10mL). Potassium hydroxide (1 g, 17.86 mmol) was added to the solution,and kept stirring under nitrogen for 4 hours. The volatile solvent wasremoved under vacuum, and the aqueous solution was extracted with ethylacetate (2×10 mL). The combined organic layers were washed with brine(3×10 mL), dried with magnesium sulfate, filtered and concentrated. Theresidue was purified with flash chromatography (2:1 hexanes:ethylacetate) to yield the alcohol (35b) as colorless oil (2.34 g, 100% yieldover 2 steps). ¹H NMR (CDCl₃) 3.96 (s, 2H), 1.63-1.76 (m, 4H), 1.10-1.18(m, 6H), −0.13-0.77 (m, 3H) ³¹P NMR (CDCl₃) 23 (q, J=56.7 Hz).

A solution of the alcohol 35b (743 mg, 5.58 mmol) and triethylamine (1.2mL, 8.6 mmol) in freshly distilled methylene chloride (10 mL) was cooledto 0° C. To this solution, mesyl chloride 37b (600 μL, 7.75 mmol) wasadded, and the mixture was stirred under nitrogen for over night. Thesolution was washed with water (2×5 mL), HCl (0.5M, 5 mL) and water (5mL). The organic layer was dried with magnesium sulfate, filtered andconcentrated. The residue was purified by flash chromatography (3:1hexanes:ethyl acetate) to yield the mesylate 38b as pale yellowish oil(794 mg, 67% yield). ¹H NMR (CDCl₃) 4.46 (d, J=1.8 Hz, 2H), 3.07 (s,3H), 1.49-1.84 (m, 4H), 1.11-1.22 (m, 6H), −0.13-0.75 (m, 3H) ³¹P NMR(CDCl₃) 26.08 (q, J=42.8 Hz).

The mesylate 38b (228 mg, 1.04 mmol) was dissolved in dry amine-freeN,N-dimethylformamide (2 mL), followed by addition of thiolacetic acid39b (85 μL, 1.19 mmol). The solution was cooled to 0° C. and cesiumcarbonate (340 mg, 1.04 mmol) was added. After stirring under nitrogenfor over night, the dark colored solution was filtered and concentrated.The residue was purified by flash chromatography (3:1 hexanes:ethylacetate) to yield the thiolacetate 10c as slightly yellowish oil (174mg, 87% yield). ¹H NMR (CDCl₃) 6.31 (d, J=6 Hz, 2H), 2.40 (s, 3H), 1.66(dq, J=10.2, 7.6 Hz, 4H), 1.14 (dt, J=16.1, 7.9 Hz, 6H), −0.04-0.86 (m,3H) ¹³C NMR 193.44, 30.23, 20.28 (d, J=112.27 Hz), 15.49 (d, J=141.00Hz), 3.66 (d, J=9.6 Hz) ³¹P NMR (CDCl₃) 25.92 (q, J=47.6 Hz).

The thiolacetate 10c (190 mg, 1 mmol) was dissolved in methanol (5 mL),followed by addition of sodium methoxide solution (1M in methanol, 1mL). The solution was stirred under nitrogen for 10 minutes. Thesolution was neutralized by pH 6.5 phosphate buffer, extracted withethyl acetate (2×5 mL). The combined organic layers were washed withbrine (2×5 mL), dried with sodium sulfate, filtered and concentrated.The crude product was dissolved in freshly distilled methylene chloride(5 mL) with Boc-Asp-OBzl (323 mg, 1 mmol), a protected amino acid. Tothis solution, catalytic DMAP (2 mg) and DCC (206 mg, 1 mmol) wereadded. The solution was kept stirring under nitrogen for 2 hours. Thevolatile solvent was removed under vacuum and the residue was purifiedby flash chromatography (3:1 hexanes:ethyl acetate) to yield thethiolester 63 as colorless oil (430 mg, 95.4% yield over two steps). ¹HNMR (CDCl₃) 7.31 (m, 5H) 5.44 (d, J=7.5 Hz, 1H), 5.14 (s, 2H), 4.57 (m,1H), 3.20 (broad s, 2H), 3.08 (d, J=6 Hz), 1.59 (qt, J=6.9 Hz, 4H), 1.40(s, 9H), 1.07 (dt, J=16.2, 8.4 Hz), −0.26-0.51 (m, 3H) ¹³C NMR (CDCl₃)234.85, 194.66, 170.24, 134.93, 128.49, 128.42, 129.19, 80.26, 67.55,50.52, 28.15, 20.07 (d, J=28 Hz), 15.34 (d, J=35.6 Hz), 6.55 (d, J=2.4Hz) ³¹P NMR (CDCl₃) 26.17 (d J=142.8 Hz).

The phosphine-borane complex 63 (170 mg, 0.373 mmol) and DABCO (84 mg,0.75 mmol) were dissolved in freshly distilled toluene. The solution washeated under argon at 80° C. for 2 hours. The solution was cooled toroom temperature and filtered through a plug of silica gel undernitrogen. The phosphinothiolester 64 was collected as an oil (131 mg,80% yield). ¹H NMR (CDCl₃) 7.35 (m, 5H), 5.44 (d, J=9.3 Hz, 1H), 5.19(s, 2H), 4.59 (m, 1H), 3.18-3.25 (m, 2H), 3.01 (d, J=5.4 Hz, 2H), 1.44(m, 13H), 1.05 (dt, J=18.6, 9.3 Hz, 6H). Due to the instability of thiscompound, no ¹³C NMR was performed.

TABLE 1 Staudinger Ligation of AcGlySCH₂PPh₂(51) and Non-Glycyl α-AzidoAads yield α-azido acid peptide (%)^(b)

AcGly(L)PheNHBn52-L 90

AcGly(D)PheNHBn52-D 93

AcGly(L)Asp(OMe)NHBn53-L 91

AcGly(D)Asp(OMe)NHBn53-D 95

AcGly(L)Ser(Bn)NHBn54-L 92

AcGly(D)Ser(Bn)NHBn54-D 99 ^(a)Reaction conditions: THF/H₂O (3:1) atroom temperature for 12 h. ^(b)Isolated yield product afler purificationby flash chromatography.

REFERENCES

(1) For recent reviews of peptide ligation methodology, see: (a) Tam, J.P.; Yu, Q.; Miao, Z. Biopolymers 1999, 51, 311-332. (b) Dawson, P. E.;Kent, S. B. H. Annu. Rev. Biochem. 2000, 69, 923-960. (c) Borgia, J. A.;Fields, G. B. Trends Biotechnol. 2000, 15, 243-251. (d) Miranda, L. P.;Alewood, P. F. Biopolymers 2000, 55, 217-226, (e) Tam, J. P.; Xu, J.;Eom, K. D. Biopolymers 2001, 60, 194-205.

(2) Wieland, T.; Bokelmann, E.; Bauer, L.; Lang, H. U.; Lau, H. LiebigsAnn. Chem. 1953, 583, 129-149. (b) Dawson, P. E.; Muir, T. W.;Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776-779.

(3) Cysteine is uncommon, comprising only 1.7% of the residues inproteins (McCaldon, P.; Argos, P. Proteins 1988, 4, 99-122). Modernpeptide synthesis is typically limited to peptides of ≦50 residues (ref1). Hence, most proteins cannot be prepared by any method that allowsfor the coupling of peptides only at cysteine residues.

(4) (a) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000,2, 1939-1941. (b) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T.; Org.Lett. 2001, 3, 9-12. For a review, see: (c) Gilbertson, S.Chemtracts—Org. Chem. 2001, 14, 524-528.

(5) (a) Staudinger, H.; Meyer, J. Helv. Chim. Acta 1919, 2, 635-646. Fora review see: (b) Gololobov, Yu. G.; Kasukhi, L. F. Tetrahedron 1992,48, 1353-1406.

(6) For examples in which the acyl donor is attached covalently to thephosphine, see: ref4 and (a) Saxon, E.; Bertozzi, C. R. Science 2000,287, 2007-2010. (b) Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. Org.Lett. 2000, 2, 2141-2143. (c) Kiick, K. L.; Saxon, E.; Tirrell, D. A.;Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 19-24.

(7) For examples in which the acyl donor is not attached to thephosphine, see: (a) Garcia, J.; Urpi, F.; Vilarrasa, J. TetrahedronLett. 1984, 25, 4841-4844. (b) Garcia, J.; Vilarrasa, J. TetrahedronLett. 1986, 27, 639-640. (c) Urpi, F.; Vilarrasa, J. Tetrahedron Lett.1986, 27, 4623-4624. (d) Bosch, I.; Romea, P.; Urpi, F.; Vilarrasa, J.Tetrahedron Lett. 1993, 34, 4671-4674. (e) Inazu, T.; Kobayashi, K.Synlett. 1993, 869-870. (f) Molina, P.; Vilaplana, M.J.Synthesis-Stuttgart 1994, 1197-1218. (g) Bosch, I.; Urpi, F.; Vilarrasa,J. J. Chem. Soc., Chem. Commun. 1995, 91-92. (h) Shalev, D.E.;Chiacchiera, S.M.; Radkowsky, A.E.; Kosower, E. M. J. Org. Chem. 1996,61,1689-1701. (I) Bosch, I.; Gonzalez, A.; Urpi, F.; Vilarrasa, J. J.Org. Chem. 1996, 61, 5638-5643. (j) Maunier, V.; Boullanger, P.; Lafont,D.LJ. Carbohydr. Chem. 1997, 16, 23 1-235. (k) Afonso, C.A.M. SyntheticCommun. 1998, 28, 261-276. (I) Tang, Z.; Pelletier, J.C. TetrahedronLett. 1998, 39, 4773-4776. (m) Ariza, X.; Urpi, F.; Viladomat, C.;Vilarrasa, J. Tetrahedron Lett. 1998, 39, 9101-9102. (n) Mizuno, M.;Muramoto, I.; Kobayashi, K.; Yaginuma, H.; lnazu, T. Synthesis-Stuttgart1999, 162,165. (o) Mizuno, M.; Haneda, K.; Iguchi, R.; Muramoto, I.;Kawakami, T.; Aimoto, S.; Yamamoto, K.; Inazu, T. J. Am. Chem. Soc.1999, 121, 284-290. (p) Boullanger, P.; Maunier, V.; Lafont, D.Carbohydr. Res. 2000, 324, 97-106. (q) Velasco, M.D.; Molina, P.;Fresneda P.M.; Sanz, M.A. Tetrahedron 2000, 56, 4079-4084. (r)Malkinson, J.P.; Falconer, R.A.; Toth, I. J. Org. Chem. 2000, 65,5249-5252. (s) Ariza, X.; Pineda, O.; Urpi, F.; Vilarrasa, J.Tetrahedron Lett. 2001, 42, 4995-4999.

(8) For information on exploiting the chirality of α-amino acids, see:Coppola, G. M.; Schuster, H. F. Asymmetric Synthesis. 1987. John Wiley &Sons, New York.

(9) Lu, W.; Qasim, M. A.; Kent, S. B. H. J. Am. Chem. Soc. 1996, 118,8518-8523.

(10) Zaloom, J.; Roberts, D. C. J. Org. Chem. 1981, 46, 5173-5176.

(11) Because the reaction products herein are enantiomers, we actuallyprobe “racemization” (Reist, M.; Testa, B.; Carrupt, P.-A.; Jung, M.;Schurig, V. Chirality 1995, 7, 396-400) We use the term “epimerization”to focus attention on the chirality of the α-carbon.

(12) For reviews of borane protection of phosphines see: (a) Brunel, J.M.; Faure, B.; Maffei, M. Coord. Chem. Rev. 1998, 180, 665-698. (b)Carboni, B.; Monnier, L. Tetrahedron 1999, 55, 1197-1248.

(13) Farrington, G. K.; Kumar, A.; Wedler, F. C. Org. Prep. Proced. Int.1989, 21, 390-392.

(14) Imamoto, T.; Oshiki, T.; Onozawa, T.; Kusumoto, T.; Sato, K. J. Am.Chem. Soc. 1990, 112, 5244-5252.

(15) Brisset, H.; Gourdel, Y.; Pellon, P.; Le Corre, M. TetrahedronLett. 1993, 34, 4523-4526.

(16) Romoff, T. T.; Goodman, M. J. Peptide Res. 1997, 49, 281-292.

(17) Lundquist, J. T., IV; Pelletier, J. C. Org. Lett. 2001, 3, 781-783.

1. A phosphine-borane complex of formula:

where R and R′, independently of one another, are optionally substitutedalkyl, aryl or alkoxy groups where R and R′ may be the same or differentgroups and R and R′ may be covalently linked to each other; R″, independently of other R″ in the compound, are selected from H, anoptionally substituted alkyl or an optionally substituted aryl groupwhere all three of R″ may be the same or each may be different, and anytwo or three of R″ may be covalently linked to each other; R² and R³,independently of one another, are selected from H, an optionallysubstituted alkyl or an optionally substituted aryl group, where R² andR³ may be covalently linked to each other; n is a integer; and AA is anamino acid, peptide or protein or a fully or partially protectedderivative thereof.
 2. The complex of claim 1 wherein the AA group is anamino acid or a fully or partially protected derivative thereof.
 3. Thecomplex of claim 1 wherein the AA group is a peptide or a fully orpartially protected derivative thereof.
 4. The complex of claim 1wherein n is 1, 2 or 3 .
 5. The complex of claim 1 wherein R and R′ areoptionally substituted alkyl, alkoxy or aryl groups.
 6. The complex ofclaim 1 wherein R and R′ are ethyl groups.
 7. The complex of claim 1wherein R², R³ are H.
 8. The complex of claim 1 R″ are all H or allsmall alky groups.
 9. A kit for forming an amide bond which comprisesone or more phoshine-borane complexes of claim 1 .
 10. The complex ofclaim 1 wherein n is 1 and AA is selected from the group consisting ofglycine, alanine, valine, leucine, isoleucine, phenylalanine, serine,methionine, proline, tyrosine, tryptophan, lysine, arginine, histidine,asparate, glutamate, asparagine, glutamine, cysteine, methionine,hydroxyproline, γ-carboxyglutamate, O-phosphoserine, ornithine,homoarginine and protected derivatives thereof.
 11. The complex of claim10 wherein n is 1 and AA is a protected derivative of an amino acidselected from the group consisting of glycine, alanine, valine, leucine,isoleucine, phenylalanine, serine, methionine, proline, tyrosine,tryptophan, lysine, arginine, histidine, asparate, glutamate,asparagine, glutamine, cysteine, methionine, hydroxyproline,γ-carboxyglutamate, O-phosphoserine, ornithine, and homoarginine. 12.The complex of claim 1 wherein n is 1 and AA is a protected amino acid.13. The complex of claim 12 wherein the AA is an N-acetyl protectedamino acid.
 14. The complex of claim 12 wherein the AA is N-acetylglycine or N-acetyl phenylalanine.
 15. The complex of claim 13 wherein Rand R′ are phenyl groups.
 16. The complex of claim 13 wherein R and R′are alkyl groups.
 17. The complex of claim 13 wherein R² and R³ are H.18. The complex of claim 16 wherein R and R′ are phenyl groups.
 19. Thecomplex of claim 17 wherein all R″ are H.
 20. The complex of claim 1wherein the AA is a side-group protected amino acid.
 21. The complex ofclaim 20 wherein the AA is a protected aspartate, glutamate, asparagine,or glutamine.
 22. The complex of claim 1 wherein the AA is an amino acidhaving a side-group carrying a carboxyl group and wherein the thioesteris formed at the carboxyl group of the amino acid side group.
 23. Thecomplex of claim 22 wherein the AA is aspartate or glutamate.
 24. Thecomplex of claim 23 which is


25. A kit for forming an amide bond which comprises one or morephoshine-borane complexes of claim 2 .
 26. The kit of claim 25 whereinin the phosphine-borane complexes n is 1 and AA is selected from thegroup consisting of glycine, alanine, valine, leucine, isoleucine,phenylalanine, serine, methionine, proline, tyrosine, tryptophan,lysine, arginine, histidine, asparate, glutamate, asparagine, glutamine,cysteine, methionine, hydroxyproline, γ-carboxyglutamate,O-phosphoserine, ornithine, homoarginine and protected derivativesthereof.
 27. The kit of claim 9 further comprising one or more azides.28. The kit of claim 27 wherein the azide is an azide of an amino acid,a peptide, a protein, a carbohydrate, a monosaccharide, a disaccharide,a trisaccharide, a nucleoside, a reporter, a tag or label.